The goal of this application is to understand the mechanisms of cardioprotection with a focus on signaling pathways, particularly those that regulate ion homeostasis during myocardial ischemia and reperfusion. This approach is based on our previous finding that an increase in cytosolic calcium is a critical regulator of ischemia/reperfusion injury. In the previous funding period, we examined several types of cardioprotection, including ischemic preconditioning and erythropoietin (EPO) induced cardioprotection, and we demonstrated male-female differences in ischemia/reperfusion injury under hypercontractile conditions. We found that cardioprotective interventions generally minimize ionic alterations during ischemia, and a common feature of many cardioprotective interventions is activation of signaling kinases. A major focus of this application is the cardioprotective effect of signaling kinases such as phosphatidylinositol-3-kinase and glycogen synthase kinase-3beta. Our previous work has demonstrated a critical role for endosomal signaling in the protective effect of ischemic preconditioning, and the first specific aim is to determine how endosomal signaling contributes to cardioprotection. We have shown a cardioprotective effect of EPO, administered prior to ischemia or at the start of reperfusion;EPO activates many of the same signaling cascades as ischemic preconditioning, and in the second specific aim, we will investigate the mechanistic basis of EPO-induced cardioprotection, including the possible role of endosomal signaling. We have also demonstrated male- female differences in ischemia/reperfusion injury, and we will continue our work to establish the mechanisms involved in the third specific aim. We have found differences in sodium ion gradients, in calcium handling by the sarcoplasmic reticulum, and in nitrosylation of proteins such as the L-type calcium channel in male versus female hearts;these differences can largely be eliminated by NOS inhibitors, and in genetically altered mouse hearts lacking a NOS isoform. We will explore the importance of S-nitrosylation and signaling pathways in the male-female differences. These studies will increase our understanding of mechanisms of injury that occur when there is insufficient blood flow to the heart (a heart attack) and mechanisms that can protect the heart from this injury, leading to better treatment options for patients with coronary artery disease and for patients undergoing heart surgery where blood flow to the heart is temporarily stopped.